The present invention relates to retro peptides, as well as antibodies thereto, and to their uses, chiefly in the field of preparation of pharmaceutical compositions, in particular vaccines, and for in vitro diagnosis of various pathologies.
The development of neuropeptides, peptide hormones and antibiotics based on peptides or of synthetic vaccines based on peptides faces great problems due to the high sensitivity of peptides to proteolysis, which limits, inter alia, oral and parenteral administration.
For several years, attention has been paid to the synthesis of peptide analogues in order to investigate peptides which mimic natural peptides or proteins and have an increased activity and longer biological half-life compared to the latter. For example, peptide analogues have been obtained by replacing the L amino acids of the natural peptide by the corresponding D amino acids, or by non-natural residues (for example sarcosine and xcex2-alanine), or also by modification of peptide bonds of the natural peptide (Chorev, M. and Goodman, M. (1993), Acc. Chem. Res. 26, 266-273; Marraud et al., (1993), Biopolymers, 33, 1135-1148).
These modifications give pseudopeptides or peptides which mimic the natural peptides or proteins (also called peptidomimetics) and have a metabolic stability which is greater than that of the latter, since the majority of natural proteases cannot cleave the D amino acids and non-peptide bonds.
The major problem encountered with such pseudopeptides is that of preserving their biological activity with respect to that of the natural peptide, or of the natural protein which they are supposed to mimic.
The D form of the protease HIV-1 has recently been synthesized (De L. Milton et al., (1992), Science, 256, 1445-1448). As was to be expected, the enantiomeric protein showed a reciprocal chiral specificity such that the enzyme was incapable of cleaving the normal L substrate, but hydrolysed its D enantiomer.
In contrast, Wen and Laursen (Wen, D. and Laursen, R. A., (1993), FEBS Lett., 317, 31-34) have shown that both the D and the L form of an xcex1-helicoidal anti-icing polypeptide bond equally well to the same substrate of achiral ice, while Wade et al. (Wade et al., (1990), Proc. Natl. Acad. Sci., USA, 87, 4761-4765) found that the L and D enantiomers of several antibiotics which form channels were all as active as one another.
The modified peptides could be used as potential synthetic vaccines if they could induce the formation of antibodies which recognize the non-modified antigenic structures of the corresponding pathogen and neutralize its infectious character.
However, in comparison with the considerable work carried out to date in the field of production of antibodies to natural proteins or to synthetic peptides derived from the latter, and the study of cross-reactions between such antibodies and these natural proteins or peptides, little is known of the immune response to peptide analogues, in particular to the D peptides and the peptides containing modified bonds.
Several authors have asserted that pseudopeptides probably have very little or no immunogenicity, since they could not be transformed and presented to molecules of major histocompatibility complex (MHC) in order to be recognized by auxiliary T cells or by cytotoxic T lymphocytes. Consequently, Dintzis et al. (8) have recently reported that the L enantiomer of rubrexodin induces a strong immune response by producing immunoglobulins of the G isotype (IgG), while the corresponding protein made up of amino acids all with the D configuration does not induce an immune response.
The object of the present invention is to provide pharmaceutical compositions, and more particularly vaccines, comprising peptide analogues which have a half-life which is clearly superior to that of natural proteins or that of synthetic peptides which are or are not derived from these natural proteins (these natural proteins, or peptides which are or are not derived from the latter, also being referred to in the following by the expression xe2x80x9cparent proteins or peptidesxe2x80x9d), of which they are the analogues, while having a comparable, or even higher, biological, and more particularly immunological, activity to that of the abovementioned parent proteins or peptides.
The object of the present invention is also to provide methods for in vitro diagnosis of diseases associated with the presence in the organism of an individual of endogenous or exogenous proteins, these methods being carried out with the aid of peptide analogues as defined above and having the advantage of being more efficient than the current methods of diagnosis carried out with the aid of the parent peptides or proteins. The object of the present invention is thus, in particular, to provide new kits for implementing such methods of diagnosis.
The present invention chiefly relates to the use
of compounds of the peptide type (also called peptide analogues, or pseudopeptides, or peptidomimetics),
in which at least one of the xe2x80x94COxe2x80x94NHxe2x80x94 bonds, and advantageously all the xe2x80x94COxe2x80x94NHxe2x80x94 bonds, of the peptide chain of the corresponding parent peptide (containing no xe2x80x94NHxe2x80x94COxe2x80x94 bond in its peptide chain) is (are) replaced by (a) xe2x80x94NHxe2x80x94COxe2x80x94 bond(s),
the chirality of each aminoacyl residue, whether involved or not in one or more of the abovementioned xe2x80x94NHxe2x80x94COxe2x80x94 bonds, being either maintained or reversed with respect to the corresponding aminoacyl residues which make up the said parent peptide,
these compounds of the peptide type also being called immunoretroids, or
antibodies to the said immunoretroids (anti-immunoretroid antibodies),
the said immunoretroids being capable of forming a complex with the said anti-immunoretroid antibodies as well as with antibodies to the parent peptides or proteins (called anti-parent antibodies), and/or to the enantiomers of these parent peptides or proteins,
for the preparation:
of a medicament intended for prevention or treatment of diseases associated with the presence in the organism of an individual of an exogenous or endogenous protein capable of being recognized by the abovementioned anti-immunoretroid or anti-parent antibodies, or
of a medicament intended for prevention or treatment of diseases involving molecules of major histocompatibility complex and/or T cell receptors,
of a medicament intended for prevention or treatment of diseases associated with the presence in the organism of an individual of an antibody to an endogenous or exogenous protein capable of being recognized by a said immunoretroid,
or for implementation of a method of in vitro diagnosis of the abovementioned diseases.
As has already been seen above, the abovementioned term xe2x80x9cparent peptidexe2x80x9d is to be understood as meaning
a peptide which exists as such in the natural state, in particular in a microorganism or in a higher organism (in particular in the human organism),
or any peptide of immunological interest obtained by peptide synthesis,
or a peptide derived from a protein such as exists in the natural state in the abovementioned organisms, in particular by fragmentation of the said protein (in particular with the aid of suitable proteases, followed by purification of the peptide in question), or by peptide synthesis (by the methods conventionally used in this field)
or a peptide derived from a protein such as exists in the natural state but of which the immunological activity has been modified, maintained or optimized by replacing certain amino acids of the natural sequence, for example following screening of a library of analogous peptides obtained by peptide synthesis.
It goes without saying that the xe2x80x94COxe2x80x94NHxe2x80x94 and xe2x80x94NHxe2x80x94COxe2x80x94 bonds should be considered in the above and the following, in the direction of the parent peptide chain from the amino-terminal (N-terminal) end towards the carboxy-terminal (C-terminal) end.
The immunoretroids used in the invention can be linear or cyclic or branched.
The immunoretroids used in the context of the present invention are compounds made up of a peptide chain in which at least one of the residues, this residue being, as appropriate, of opposite chirality to that of the aminoacyl radical corresponding to it in the peptide chain of the parent peptide, is bonded to at least one of its neighbouring residues by an xe2x80x94NHxe2x80x94COxe2x80x94 bond, the said peptide chain containing, as appropriate, one or more aminoacyls of opposite chirality to that of the aminoacyl residue corresponding to it in the peptide chain of the parent peptide, it being possible for the amino- and carboxy-terminal ends, independently of one another, to be either identical to the N- and C-terminal ends of the corresponding parent peptide or different from these latter ends.
A residue is understood as meaning a group of the formula xe2x80x94Xxe2x80x94CH(R)xe2x80x94Yxe2x80x94, in which X and Y are identical to or different from one another and are chosen from xe2x80x94NHxe2x80x94 or xe2x80x94COxe2x80x94. Hence either an aminoacyl residue or an aminoacyl derivative in which the terminal ends are not those of an aminoacyl residue are thus called residues.
By way of illustration, the immunoretroids used in the context of the present invention are those derived from parent peptides which correspond to the following formula (I):
Xxe2x80x94AA1xe2x80x94(AA2xe2x80x94xe2x80x94xe2x80x94xe2x80x94xe2x80x94AAnxe2x88x921)ixe2x80x94AAnxe2x80x94Y xe2x80x83xe2x80x83(I) 
in which:
AA1 represents an aminoacyl residue, which may be deaminated and in which the amine function in the xcex1-position, if this exists, can be protected by a grouping X, X representing Pxe2x80x94, Rxe2x80x94 or RCOxe2x80x94,
i=0 or 1,
n represents 2 if i=0, and n represents an integer from 3 to 1,000, and preferably from 5 to 100, if i=1, it being understood that if n=3, the corresponding immunoretroid corresponds to the formula AA1xe2x80x94AA2xe2x80x94AA3,
AAn represents an aminoacyl residue which may be decarboxylated and in which the acid function in the xcex1-position, if this exists, is optionally protected by a grouping Y, Y being of the ester xe2x80x94OR or amide xe2x80x94NH2 or xe2x80x94NRRxe2x80x2 type,
it being possible for the groupings R and Rxe2x80x2 to represent hydrogen atoms, alkyl radicals having 1 to 25 carbon atoms, radicals containing an allyl group and having 3 to 25 carbon atoms or radicals containing an aryl group and having 6 to 25 carbon atoms, and in particular xe2x80x94CH3 (methyl), xe2x80x94CH2CH3 (ethyl), xe2x80x94CH(CH3)2 (isopropyl), xe2x80x94C(CH3)3 (tert-butyl), xe2x80x94"PHgr" (phenyl), xe2x80x94CH2"PHgr" (benzyl), xe2x80x94CH2xe2x80x94CH2"PHgr" (2-phenyl-ethyl), xe2x80x94CH2CHCH2 (allyl), methyl-fluorenyl, xe2x80x94CH2CONH2 (glycolamide), or xe2x80x94CH2CON"PHgr"2 (benzhydrylglycolamide), this list not being limiting,
the grouping P being of the urethane type (Boc (tert-butyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), Z (benzyloxycarbonyl), CH2CHCH2OCO-(allyloxycarbonyl) or other),
and in these parent peptides of the formula (I), at least one of the bonds between two aminoacyl residues of the formula I being an xe2x80x94NHxe2x80x94COxe2x80x94 bond, and, as appropriate, at least one of the aminoacyl residues AA1 to AAn being of opposite chirality to that of the corresponding aminoacyl residue in the parent peptide.
In the peptides used in the context of the present invention, the xe2x80x94NHxe2x80x94COxe2x80x94 bonds which replace the xe2x80x94COxe2x80x94NHxe2x80x94 bonds of the corresponding parent peptide can be in any position in the peptide, and can replace either a xe2x80x94COxe2x80x94NHxe2x80x94 corresponding to a cleavage site for proteases or a xe2x80x94COxe2x80x94NHxe2x80x94 which does not correspond to such a site.
The xe2x80x94NHxe2x80x94COxe2x80x94 bonds which replace the xe2x80x94COxe2x80x94NHxe2x80x94 bonds are advantageously those which interact with the peptide receptor (antibody, MHC, T receptor).
According to an advantageous embodiment of the invention, the immunoretroids preferably correspond to the following formula (II):
Axe2x80x94CH(Ri)xe2x80x94NHxe2x80x94[COxe2x80x94CH(Rk)xe2x80x94NH]jxe2x88x92ixe2x80x94COxe2x80x94CH(Rj+1)xe2x80x94B xe2x80x83xe2x80x83(II) 
in which
Ri, Rk and Rj+1 represent side chains of the residues involved in one or more xe2x80x94NHxe2x80x94COxe2x80x94 bonds,
the total number of residues of the sequence is fixed at n, where n is an integer greater than 1, and preferably from 3 to 1,000, preferably 3 to 100,
i, j and k are whole parameters defined in the following manner:
1xe2x89xa6i less than jxe2x89xa6n.
k=0 if i=j and k assumes the values i+1 to j,
where four cases of the figure can be given regarding the nature of blocks A and B:
1/ i=1 and j+1=n:
A=Hxe2x80x94, H2Nxe2x80x94, Pxe2x80x94HNxe2x80x94, RRxe2x80x2Nxe2x80x94, H2NCOxe2x80x94, RRxe2x80x2NCOxe2x80x94, RCOxe2x80x94
B=Hxe2x80x94, xe2x80x94COOH, xe2x80x94COOR, xe2x80x94CONH2, xe2x80x94CONRRxe2x80x2, xe2x80x94NHCOR. 
2/ i=1 and j+1xe2x89xa0n:
A=Hxe2x80x94, H2Nxe2x80x94, Pxe2x80x94HNxe2x80x94, RRxe2x80x2Nxe2x80x94, H2NCOxe2x80x94, RRxe2x80x2NCOxe2x80x94, RCOxe2x80x94
B=xe2x80x94COxe2x80x94NHxe2x80x94CH(Rj+2)xe2x80x94COxe2x80x94 . . . xe2x80x94NHxe2x80x94CH(Rn)xe2x80x94COxe2x80x94Y 
where Y=xe2x80x94OH, xe2x80x94OR, xe2x80x94NH2,, xe2x80x94NRRxe2x80x2.
3/ ixe2x89xa01 and j+1=n:
A=Xxe2x80x94HNxe2x80x94CH(R1)xe2x80x94COxe2x80x94 . . . xe2x80x94NHxe2x80x94CH(Rixe2x88x921)COxe2x80x94NHxe2x80x94
where X=Hxe2x80x94, Pxe2x80x94, Rxe2x80x94, RCOxe2x80x94
B=Hxe2x80x94, xe2x80x94COOH, xe2x80x94COOR, xe2x80x94CONH2, xe2x80x94CONRRxe2x80x2, xe2x80x94NHCOR. 
4/ ixe2x89xa01 and j+1xe2x89xa0n:
A=Xxe2x80x94HNxe2x80x94CH(R1)xe2x80x94COxe2x80x94 . . . xe2x80x94NHxe2x80x94CH(Rixe2x88x921)COxe2x80x94NHxe2x80x94
where X=Hxe2x80x94, Pxe2x80x94, Rxe2x80x94, RCOxe2x80x94
B=xe2x80x94COxe2x80x94NHxe2x80x94CH(Rj+2)xe2x80x94COxe2x80x94 . . . xe2x80x94NHxe2x80x94CH(Rn)xe2x80x94COxe2x80x94Y 
where Y=xe2x80x94OH, xe2x80x94OR, xe2x80x94NH2, xe2x80x94NRRxe2x80x2,
it being possible for the groupings R and Rxe2x80x2 to represent hydrogen atoms, alkyl radicals having 1 to 25 carbon atoms, radicals containing an allyl group and having 3 to 25 carbon atoms or radicals containing an aryl group and having 6 to 25 carbon atoms, and in particular xe2x80x94CH3 (methyl), xe2x80x94CH2CH3 (ethyl), xe2x80x94CH(CH3)2 (isopropyl), xe2x80x94C(CH3)3 (tert-butyl), xe2x80x94"PHgr" (phenyl), xe2x80x94CH2"PHgr" (benzyl), xe2x80x94CH2xe2x80x94CH2"PHgr" (2-phenyl-ethyl), xe2x80x94CH2CHCH2 (allyl), methyl-fluorenyl, xe2x80x94CH2CONH2 (glycolamide) or xe2x80x94CH2CON"PHgr"2 (benzhydrylglycolamide), this list not being limiting,
the grouping P being of the urethane type (Boc (tert-butyloxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), Z (benzyloxycarbonyl), CH2CHCH2OCOxe2x80x94 (allyloxycarbonyl) or other),
the chirality of reach residue, whether involved or not in one or more xe2x80x94NHxe2x80x94COxe2x80x94 bonds, being either maintained or reversed with respect to the corresponding aminoacyl residues which make up the parent peptide.
If i=1 and j+1=n, all the bonds are xe2x80x94NHxe2x80x94COxe2x80x94 bonds.
If i=1 and j+1xe2x89xa0n, the xe2x80x94NHxe2x80x94COxe2x80x94 bonds are on the N-terminal side.
If ixe2x89xa01 and j+1xe2x89xa0n, the xe2x80x94NHxe2x80x94COxe2x80x94 bonds are on the C-terminal side.
If ixe2x89xa01 and j+1xe2x89xa0n, the xe2x80x94NHxe2x80x94COxe2x80x94 bonds are on neither the N-terminal side nor the C-terminal side.
The number of xe2x80x94NHxe2x80x94COxe2x80x94 bonds is equal to jxe2x88x92i+1.
The number of xe2x80x94NHxe2x80x94COxe2x80x94 bonds is advantageously at least equal to 2.
The invention more particularly relates to the abovementioned use of retro-inverso peptides as immunoretroids.
Retro-inverso peptides are to be understood as meaning any peptide and peptide analogue corresponding to the definition given above for immunoretroids used in the context of the present invention, the said peptide being more particularly made up of a peptide chain in which at least one of the residues on the one hand is bonded to at least one neighbouring residue by an xe2x80x94NHxe2x80x94COxe2x80x94 bond and on the other hand, if the residue is an aminoacyl residue, is of opposite chirality to that of the same aminoacyl residue in the peptide chain of the parent peptide.
The retro-inverso peptides used in the context of the present invention are more particularly those as defined above, and correspond to the abovementioned formula (II), in which at least one of the residues is bonded to at least one of its neighbouring residues by an xe2x80x94NHxe2x80x94COxe2x80x94 bond, and if the residue is an aminoacyl residue, is of opposite chirality to that of the corresponding aminoacyl residue in the parent peptide.
These abovementioned retro-inverso peptides are:
partly retro-inverso peptides, that is to say:
a) either partly retro-totally inverso peptides, that is to say in which only one or more of, but not all, the bonds between the residues is (are) (an) xe2x80x94NHxe2x80x94COxe2x80x94 bond(s), and in which all the aminoacyl residues bonded to at least one neighbouring residue by an xe2x80x94NHxe2x80x94COxe2x80x94 bond is (are) of opposite chirality to that of the corresponding aminoacyl residue in the parent peptide,
b) or partly retro-partly inverso peptides, that is to say in which only one or more of, but not all, the bonds between the residues is (are) (an) xe2x80x94NHxe2x80x94COxe2x80x94 bond(s), and in which at least one of, but not all, the aminoacyl residues bonded to at least one neighbouring residue by an xe2x80x94NHxe2x80x94COxe2x80x94 bond is (are) of opposite chirality to that of the corresponding aminoacyl residue in the parent peptide, these two types of partly retro-inverso peptides being represented by the formula (II), and more particularly is described by the cases of FIGS. 2, 3 and 4 which relate to them,
or totally retro-partly inverso peptides, that is to say in which all the bonds between the residues are xe2x80x94NHxe2x80x94COxe2x80x94 bonds, and in which at least one of, but not all, the aminoacyl residues bonded to at least one neighbouring residue by an xe2x80x94NHxe2x80x94COxe2x80x94 bond is (are) of opposite chirality to that of the corresponding aminoacyl residue in the parent peptide,
or totally retro-inverso peptides, that is to say in which all the bonds between the residues are xe2x80x94NHxe2x80x94COxe2x80x94 bonds, and in which all the aminoacyl residues of the peptide chain of these retro-inverso peptides are of opposite chirality to that of their corresponding aminoacyl residues in the parent peptide.
These last two types of retro-inverso peptides are represented by the formula (II), and are more particularly described by the case of FIG. 1 which relates to them.
The retro-inverso peptides used in the context of the present invention are advantageously totally retro-inverso.
Another advantageous class of retro-inverso peptides used in the context of the invention has at least two consecutive xe2x80x9cretro-inversoxe2x80x9d bonds.
The invention more particularly relates to the abovementioned use of retro peptides as immunoretroids.
Retro peptide is to be understood as meaning any peptide corresponding to the definition given above for immunoretroids used in the context of the present invention, the said peptide being more particularly made up of a peptide chain in which at least one of the residues is bonded to at least one neighbouring residue by an xe2x80x94NHxe2x80x94COxe2x80x94 bond, the chirality of all the aminoacyl residues involved in at least one xe2x80x94NHxe2x80x94COxe2x80x94 bond being maintained with respect to the corresponding residue of the peptide chain of the parent peptide.
The retro peptides used in the context of the present invention are more particularly those such as are defined above and correspond to the abovementioned formula (II), in which at least one of the residues is bonded to at least one neighbouring residue by an xe2x80x94NHxe2x80x94COxe2x80x94 bond.
The abovementioned retro peptides are:
either partly retro peptides, represented by the formula (II), and more particularly described by the cases of FIGS. 2, 3 and 4 which relate to them, that is to say in which only one or more of, but not all, the bonds between the aminoacyl residues is (are) (an) xe2x80x94NHxe2x80x94COxe2x80x94 bond(s),
or totally retro peptides, represented by the formula (II), and more particularly described by the case of FIG. 1 which relates to them, that is to say in which all the bonds between the aminoacyl residues are xe2x80x94NHxe2x80x94COxe2x80x94 bonds.
The totally retro peptides used in the context of the present invention are advantageously totally retro.
Another suitable class of retro peptides used in the invention have two consecutive xe2x80x9cretroxe2x80x9d bonds.
The diseases which can be diagnosed or treated with the aid of pharmaceutical compositions based on immunoretroids in the context of the present invention are chiefly diseases of a viral or bacterial origin, or are autoimmune diseases, or also neurodegenerative diseases.
Among the diseases of viral origin which can be diagnosed or treated in the context of the present invention there may be mentioned:
AIDS caused by the human immunodeficiency virus HIV1 and HIV2,
paraplegia associated with HTVL-1, or adult T cell leukaemia caused by the human T cell leukaemia virus (HTLV virus),
infections caused by the respiratory syncytial virus,
infections caused by the Coxsackie virus, for example acute lymphocytic meningitis,
infections caused by the Epstein-Barr virus, for example infectious mononucleosis,
infections caused by the cytomegalovirus, for example cytomegalic inclusion disease,
herpes caused by the human herpes virus,
herpes caused by the herpes simplex 6 virus,
infections caused by the human Parvovirus B19, for example infectious gastroenteritis,
hepatitis B caused by the hepatitis B virus,
hepatitis C, caused by the hepatitis C virus,
influenza caused by the influenza virus,
rubella caused by the rubeola virus,
infections caused by the Dengue virus, for example arboviroses,
colds, rhinitis or coryza caused by rhinoviruses and
foot-and-mouth disease caused by the foot-and-mouth disease virus.
Among the main autoimmune diseases which can be treated in the context of the present invention there may be mentioned those summarized in Table A which follows.
The invention also relates to antibodies to the immunoretroids according to the invention, also called anti-immunoretroid antibodies.
The anti-immunoretroid antibodies according to the invention are polyclonal or monoclonal antibodies.
The abovementioned polyclonal antibodies are obtained by immunization of an animal with at least one immunoretroid according to the invention, followed by recovery of the required antibodies in a purified form by taking serum from the said animal and separating the said antibodies from others contained in the serum, in particular by affinity chromatography over a column on which is fixed an antigen recognized specifically by the antibodies, in particular an immunoretroid according to the invention.
The monoclonal antibodies according to the invention can be obtained by the hybridoma technique, the general principle of which is described below.
An animal, generally a mouse (or cells in culture in the context of in vitro immunizations) is first immunized with an immunoretroid according to the invention, the B lymphocytes of which then being capable of producing antibodies to the immunoretroid and/or to this protein and/or to the parent peptide. These lymphocytes which produce antibodies are then fused with xe2x80x9cimmortalxe2x80x9d myelomatous cells (murine cells in the example) to give rise to hybridomas. From the heterogeneous mixture of cells thus obtained, cells capable of producing a particular antibody and of multiplying indefinitely are then selected. Each hybridoma is multiplied in the form of a clone, each leading to the production of a monoclonal antibody of which the recognition properties with respect to the immunoretroid of the invention can be tested, for example, by ELISA, by immunotransfer in one or two dimensions, under immunofluorescence or with the aid of a biocaptor. The monoclonal antibodies selected in this way are then purified, in particular by the technique of affinity chromatography described above.
The antibodies according to the invention are more particularly characterized in that they are capable of forming a complex with the immunoretroids, and/or with the parent peptides or proteins corresponding to the latter.
In this respect, the invention more particularly relates to the antibodies as defined above, which are characterized in that they recognize the retro-inverso peptides and/or the corresponding parent peptides or proteins, in particular the parent peptides or proteins of the L configuration.
The invention also more particularly relates to antibodies as defined above, which are characterized in that they recognize the retro peptides, and/or the enantiomers of the corresponding parent peptides or proteins, in particular the enantiomers of the parent peptides or proteins of the L-configuration.
The antibodies according to the invention are more particularly also characterized in that they are protective antibodies, that is to say antibodies which, if they are administered into the organism of an individual carrying a pathogenic endogenous or exogenous protein or if their formation is induced by administration into the organism of the individual of an immunoretroid according to the invention which can be recognized by such antibodies, are capable of bonding to the said endogenous or exogenous protein under conditions such that the pathogenic character associated with this protein is thus neutralized.
The anti-immunoretroid antibodies of the invention recognize the parent peptide or the parent protein with an affinity which is at least equal to that shown by the anti-parent peptide or anti-parent protein antibodies with respect to the parent peptide or the parent protein.
The anti-parent peptide or anti-parent protein antibodies recognize the immunoretroids used in the context of the invention with an affinity which is at least equal to that shown by these same said antibodies with respect to the parent peptides or parent proteins corresponding to these said immunoretroids.
The affinity referred to above can be measured by the equilibrium affinity constant Ka of complexes involving one of the said antibodies with one of the said antigens.
The invention also relates to anti-idiotypes which are capable of forming a complex with the antibodies according to the invention, these anti-idiotypes being obtained by immunization of an animal with the said antibodies as defined above according to the invention.
The invention also more particularly relates to antigen-antibody complexes formed between the immunoretroids according to the invention, or their parent peptide or parent protein, and the antibodies as defined above.
In this respect, the invention more particularly relates to the following complexes:
Particularly preferred antigen-antibody complexes in the context of the invention are the following:
Parent peptide derived from the parent protein means a sequence of the parent protein.
It should be noted that all the complexes defined above are such that they have a stability which is at least equal to that of these complexes in which, if an immunoretroid occurs, it is replaced by a parent peptide or a parent protein, and if an anti-immunoretroid antibody occurs, it is replaced by an anti-parent peptide or anti-parent protein antibody.
In particular, the parent peptide/parent protein-anti-immunoretroid antibody complexes are at least as stable as the complexes of parent peptide/parent protein-antibody to the parent peptide or protein corresponding to these said immunoretroids.
It should also be noted that the complexes of immunoretroid-antibody to the parent peptide or the parent protein are at least as stable as the corresponding complexes in which the immunoretroid peptides are replaced by the parent peptide or the parent protein.
The invention advantageously relates to antigen-antibody complexes in which the antigen is an immunoretroid and/or the antibody is an antibody to an immunoretroid and which have an affinity constant greater than the affinity constant shown by the parent antigen (that is to say parent peptide or parent protein)-antibody complexes with respect to the parent antigens (that is to say antibodies to the parent peptide or the parent protein).
According to an advantageous embodiment, the invention relates to the antigen-antibody complexes in which the antigen is an immunoretroid (for example a retro-inverso) and the antibody is an antibody to the parent peptide or the parent protein (corresponding to the said retro-inverso) and which have an affinity constant greater than the affinity constant shown by the antigen-antibody complex in which the antigen is the parent peptide or the parent protein and the antibody is an antibody to the parent peptide or the parent protein.
In order to define the concepts, the order of magnitude according to which xe2x80x9cthe affinity constant is greaterxe2x80x9d may vary from about 5 to about 1,000, in particular from about 7 to about 1,000, advantageously from about 10 to 1,000, and in particular from about 10 to about 100.
The invention also relates to the complexes between an immunoretroid as defined above and a molecule of major histocompatibility complex (also called MHC-immunoretroid complex).
In fact, the immune response involves recognition of an endogenous or exogenous antigen by specialized cells. To be recognized, the antigen should initially be presented in an adequate manner by antigen-presenting cells (APC). Whereas B lymphocytes recognize epitopes carried by intact non-modified antigens, presentation of the antigen to T lymphocytes is more complex insofar as the antigen is first internalized by the presenting cell, proteolysed, and then possibly reexpressed on its surface in the form of peptide fragments in combination with the proteins of major histocompatibility complex (MHC). The T lymphocyte, which does not recognize the native antigen, recognizes a peptide fragment combined with an MHC molecule.
These MHC molecules belong to two classes: I and II.
The molecules of class I are transmembrane glycoproteins made up of a heavy polymorphic xcex1-chain combined non-covalently with a with a non-glycosylated light xcex22m chain. Their crystallographic structure has been resolved (Bjorkman et al. (1987), Nature, 329: 506-512), and shows the presence of a furrow which forms the presentation site of the peptide, the base of which is made up of eight xcex2-sheets and the sides of which are made up of two xcex1-helices. These molecules are presented on the surface of virtually all cells.
The molecules of class II are also membrane glycoproteins made up of two polymorphic xcex1- and xcex2-chains bonded non-covalently to form, as the recently elucidated crystallographic structure shows (Brown et al. (1993), Nature, 364: 33-39), a xcex2-pleated platform supporting two xcex1-helices. The furrow formed is the presentation site of the peptide. These molecules are expressed only on the surface of certain cells, including macrophages and B cells.
Cytotoxic T lymphocytes (cells which have CD8 markers) recognize proteolytic fragments of viral proteins associated with MHC molecules of class I and cause lysis of cells presenting the antigen.
Auxiliary T lymphocytes (cells which carry CD4 markers) recognize exogenous protein fragments captured by endocytosis and present in combination with the MHC molecules of class II, and induce cellular stimulation of the immune response.
The invention also relates to the complexes between an immunoretroid according to the invention and a T cell receptor.
The invention also relates to the complexes between a molecule of major histocompatibility complex, an immunoretroid as defined above, and a T cell receptor (also called MHC-immunoretroid-T receptor complex).
The invention also relates to the use of the immunoretroids as defined above for implementation of methods for in vitro diagnosis of diseases, such as those mentioned above, associated with the presence in the organism of an individual of one or more exogenous or endogenous protein(s) capable on the one hand of being involved directly or indirectly in the process of the appearance and/or development of these diseases, and on the other hand of being recognized by the anti-immunoretroid antibodies according to the invention, or by the immunoretroids according to the invention, for example in the case of detection of antibodies in the patient.
In this respect, the invention more particularly relates to any method for in vitro diagnosis as defined above and comprising:
bringing a biological sample originating from a patient who may carry antibodies to the said endogenous or exogenous proteins into contact with an immunoretroid according to the invention under conditions which allow reaction between the antibodies to the said proteins, which antibodies may be present in the biological sample, and the said immunoretroid;
in vitro detection of the antigen-antibody complex as defined above according to the invention, which may be formed in the preceding stage, or
in vitro detection of the antibody in the patient by a competition test using an anti-immunoretroid antibody.
The immunoretroid used in the abovementioned method for in vitro diagnosis is advantageously a retro-inverso peptide corresponding to all or part of the said endogenous or exogenous proteins, or corresponding to a peptide which is capable of being recognized by antibodies which themselves recognize the exogenous or endogenous proteins.
According to another preferred embodiment of the abovementioned method for in vitro diagnosis, the immunoretroid used is a retro peptide corresponding to all or part of the said endogenous or exogenous proteins, or corresponding to a peptide which is capable of being recognized by antibodies which themselves recognize the exogenous or endogenous proteins.
The invention also relates to any method for in vitro diagnosis as defined above and comprising:
bringing a biological sample originating from an individual who may be a carrier of the said endogenous or exogenous proteins into contact with at least one of the antibodies, as defined above, to an immunoretroid according to the invention under conditions which allow reaction between the said proteins which may be present in the biological sample and the said antibodies to the said immunoretroid;
in vitro detection of the antigen-antibody complex as defined above which may be formed in the preceding stage, or
detection of circulating antigens in competition tests using one of the said immunoretroids.
The antibodies to the immunoretroid which are used in the abovementioned method for in vitro diagnosis are advantageously those antibodies, described above according to the invention, to a retro-inverso peptide corresponding to all or part of the said endogenous or exogenous proteins.
According to another preferred embodiment of the abovementioned method for in vitro diagnosis, the antibodies to the immunoretroid are those antibodies, as defined above according to the invention, to a retro peptide corresponding to all or part of the said endogenous or exogenous proteins.
The abovementioned methods for diagnosis of the invention are advantageously carried out in the following manner:
incubation of the biological sample which may contain the antigens (exogenous or endogenous protein or peptide), or antibodies to these antigens, these antigens or antibodies being associated with a disease or a family of specific diseases, in particular with the diseases described above, with, respectively, anti-immunoretroid antibodies according to the invention which are capable of recognizing the said antigens, or immunoretroids according to the invention which are capable of being recognized by the said antibodies to these antigens, the said antigens or antibodies to the latter being fixed on a solid support, in particular inside the wells of microtitration plates of the type usually used for implementation of detection or assay techniques commonly known by the name ELISA (enzyme-linked immunosorbent assay),
rinsing of the solid support,
incubation of the elements remaining fixed on the solid support after the preceding rinsing stage:
either with a medium comprising antibodies, in particular anti-immunoretroid antibodies according to the invention, which are marked (in particular in a radioactive, enzymatic or fluorescent manner) or are capable of being recognized in their turn by a marked reagent, the said marked antibodies being capable of recognizing the antigens present in the biological sample which remain bonded, after the preceding rinsing stage, to the anti-immunoretroid antibodies according to the invention initially fixed on the solid support,
or with a medium comprising antigens, in particular immunoretroids according to the invention, which are marked (in particular in a radioactive, enzymatic or fluorescent manner) or capable of being recognized in their turn by a marked reagent, the said marked antigens being capable of recognizing the antibodies present in the biological sample which remain bonded, after the preceding rinsing stage, to the immunoretroids according to the invention initially fixed to the solid support,
rinsing of the solid support,
detection of the marked antigens or antibodies remaining bonded, respectively, to the antibodies or antigens of the biological sample during the preceding incubation stage.
The methods for in vitro diagnosis of the invention are advantageously carried out with the aid of totally retro-inverso peptides or totally retro peptides, or also antibodies to these totally retro-inverso or totally retro peptides.
The invention also relates to the requisites or kits for implementation of the methods for in vitro diagnosis as described above, comprising:
an immunoretroid according to the invention chosen from a retro-inverso peptide and/or a retro peptide corresponding to all or part of the said endogenous or exogenous proteins, or corresponding to a peptide which is capable of being recognized by antibodies which themselves recognize the exogenous or endogenous proteins, or
anti-immunoretroid antibodies, according to the invention, to this retro-inverso peptide and/or this retro peptide;
reagents for rendering a medium capable of the formation of an immunological reaction;
reagents which allow detection of the antigen-antibody complex, belonging to the list of compounds defined above, which has been produced as a result of the immunological reaction, the said reagents optionally comprising a marker or being capable of being recognized in their turn by a marked reagent, more particularly in the case where the abovementioned immunoretroid or anti-immunoretroid antibodies are not marked.
The invention also relates to the use of at least one immunoretroid as defined above for the preparation of a medicament intended for prevention or treatment of diseases associated with the presence in the organism of an individual of one or more exogenous or endogenous protein(s) which may be directly or indirectly involved in the process of the appearance and/or development of these diseases.
The abovementioned diseases which are capable of being treated in the context of the present invention are chiefly either diseases of viral, bacterial or parasitic origin, if they are associated with the presence of the microorganism itself, or of an exogenous protein or peptide originating from a viral, bacterial or parasitic particle, or autoimmune diseases, if they are associated with the presence of endogenous proteins or peptides which disturb normal physiological functioning of an organism where the latter play an antibody role directly or induce the formation of antibodies which recognize and alter particular sites of the organism (for example by forming depots of antibody-antigen complexes, causing inflammatory states etc.).
The abovementioned pathologies can also be neurodegenerative diseases if they are associated with the presence in the organism of exogenous proteins which have the effect of causing neurological lesions.
The immunoretroids used for the preparation of pharmaceutical compositions or vaccines above and below are advantageously totally retro-inverso peptides or totally retro peptides.
The invention more particularly relates to the use of at least one immunoretroid as defined above for the preparation of a vaccine in the context of prevention of diseases associated with the presence in the organism of an individual of one or more exogenous or endogenous protein(s) capable of being recognized by antibodies to the immunoretroids or to the anti-idiotypes according to the invention.
The invention also relates to the pharmaceutical compositions, in particular vaccines, comprising at least one anti-idiotype as defined above, in combination with a physiologically acceptable vehicle.
The anti-idiotypes used in the abovementioned pharmaceutical compositions are advantageously obtained with the aid of totally retro-inverso or totally retro peptides.
The invention also relates to any pharmaceutical composition comprising at least one immunoretroid as defined above or at least one abovementioned anti-idiotype combined with a molecule, which may or may not be a protein carrier and can induce in vivo the production of antibodies which neutralize the said exogenous or endogenous proteins responsible for the disease, or induce in vivo a cytotoxic cell immune response as described above.
By way of illustration, in the field of vaccination the invention more particularly relates to the two totally retro-partly inverso peptides of the following formula:
HOxe2x80x94m(R,S)Leu-DGln-DArg-DAla-DVal-DArg-DX1-DAla-DLeu-DSer-Gly-DX2-DAsp-Gly-DArg-DVal-Gly-DSer-Gly-[Cys-NH2]
X1=Ser or Phe
X2=Leu or Pro
corresponding to the peptides [A] and [USA] derived from the major antigenic determinant situated on protein VP1 of the foot-and-mouth disease virus, and corresponding to the following formulae:
[A] [H-Cys]-Gly-Ser-Gly-Val-Arg-Gly-Asp-Ser-Gly-Ser-Ala-Leu-Arg-Val-Ala-Arg-Gln-Leu-OH xe2x80x83xe2x80x83(SEQ ID NO.1) 
[USA] [H-Cys]-Gly-Ser-Gly-Val-Arg-Gly-Asp-Phe-Gly-Ser-Ala-Pro-Arg-Val-Ala-Arg-Gln-Leu-OH xe2x80x83xe2x80x83(SEQ ID NO.2) 
The invention also more particularly relates to the totally retro-partly inverso cyclic peptide of the following formula 
X=D-xcex1, xcex2-diaminopropionic acid,
corresponding to residues 139-147 of site A of the haemagglutinin of the influenza virus (influenza strain X31), and corresponding to the following formula: 
The invention also relates to any pharmaceutical composition comprising the anti-immunoretroid antibodies according to the invention, optionally in combination with a physiologically acceptable vehicle.
This last category of pharmaceutical composition is more particularly intended for treatment of diseases associated with the presence in the organism of an individual of exogenous or endogenous proteins capable of being recognized by the anti-immunoretroid antibodies according to the invention, these latter acting as protective antibodies which neutralize the said exogenous or endogenous proteins.
The invention also relates to pharmaceutical compositions comprising, as the active substance, an antibody to a D peptide corresponding to a parent L peptide.
The invention also relates to the use of antibody to a D peptide corresponding to a parent L peptide for the preparation of an immunogenic medicament, in particular for the preparation of a vaccine.
The invention also relates to the immunoretroids as defined above, which correspond to cytotoxic or auxiliary T epitopes, or also to peptides recognized by MHC molecules of type I or II which can be used either in the preparation of synthetic vaccines or in the prevention or treatment of autoimmune diseases.
As regards their use as vaccines, the invention more particularly relates to the partly retro-inverso immunoretroids of a cytotoxic T epitope (minimum epitope 56-68: M56-68) of the matrix of the influenza virus and an auxiliary T epitope (parent sequence 830-844: TT830-844) of the tetanus toxin, the sequences of which are shown below:
1/ Sequence of M56-68:
H-Gly-Ile-Leu-Gly-Phe-Val-Phe-Thr-Leu-OH xe2x80x83xe2x80x83(SEQ ID NO.3) 
Sequence of the retro analogue:
HO-m(R,S)Leu(D)-Thr(D)Phe-(D)Val-(D)Phe-(D)Phe-Gly-(D)Leu-gIle-Gly-H 
2/ Sequence of TT830-844:
H-Gln-Tyr-Ile-Lys-Ala-Asn-Ser-Lys-Phe-Ile-Gly-Ile-Thr-Glu-Leu-OH xe2x80x83xe2x80x83(SEQ ID NO.4) 
Sequence of the retro analogue
HO-m(R,S)Leu-(D)Thr-Gly-(D)Ile-(D)Phe(D)Lys-(D)Ser-(D)Asn-(D)Ala-(D)Lys-(D)Ile-gTyr-Gln-H 
The retro-inverso analogue of M56-68 interacts with MHC I molecules and is presented to cytotoxic T lymphocytes in order to induce a cytotoxic response. The retro-inverso analogue of TT830-844 interacts with the MHC II molecules and is presented to auxiliary T lymphocytes in order to stimulate proliferation of T cells.
As regards the use of immunoretroids in the context of the preparation of medicaments intended for treatment of autoimmune diseases, it is appropriate to note that the pathogenesis of many autoimmune diseases involves presentation of autoantigens (bonded to MHC molecules) to the receptor of autoreactive T cells (TCR), which have in one way or another escaped from the tolerance process of the being. Furthermore, the development of new strategies to modulate the response of autoreactive T cells could lead to therapeutic approaches which can be used for treatment of certain autoimmune diseases.
Certain autoimmune diseases are associated with specific MHC I or II alleles. The use of blocking peptides which are capable of interaction with a given MHC molecule (for example an MHC molecule of class II associated with a particular autoimmune disease) but which cannot activate the pathogenic T cell response is thus attractive. However, the rapid degradation of the peptides studied in biological media renders their use difficult. In this case, the immunoretroids would be very advantageous because of their stability.
The phenomenon of TCR antagonism by peptide analogues to T epitopes has recently been demonstrated and the use of specific TCR antagonists has been described (De Magistris, M. T., Alexander, J., Coggeshall, M., Altmon, A., Gaeta F. C. A., Grey, H. M. and Sette, A. (1992), Cell 68: 625). These peptides are capable of inhibiting the proliferation of T cells induced by an antigen. Such antagonist peptides are obtained by substitution of one amino acid among the residues of the antigen peptide in contact with the T cell, or by incorporation of residues in contact with the TCR into a sequence of poly-alanines. The present invention relates precisely to the use of immunoretroids corresponding to antagonist peptides of TCR to obtain medicaments intended for treatment of autoimmune diseases such as those described above.
The immunoretroids can be prepared as indicated below:
By way of illustration, general methods for the synthesis of totally or partly retro-inverso or retro peptides, with or without modification of the N- and C-terminal ends with respect to the ends of the parent peptides, are indicated below.
The process for the synthesis of peptides in which all the xe2x80x94COxe2x80x94NHxe2x80x94 bonds are replaced by xe2x80x94NHxe2x80x94COxe2x80x94 bonds, in comparison with the parent peptides, without modification of the ends is carried out by connecting between them the various aminoacyl residues which make up the parent peptide in the reverse direction to that indicated for the parent peptide. Thus, if the parent peptide is represented by a sequence of the type
Hxe2x80x94AA1xe2x80x94AA2xe2x80x94 . . . xe2x80x94AAnxe2x88x921AAnxe2x80x94OH, 
the corresponding retro analogue synthesized according to the conventional techniques of peptide synthesis in the liquid or solid phase will have the following formula:
Hxe2x80x94AAnxe2x80x94AAnxe2x88x921 . . . xe2x80x94AA2xe2x80x94AA1xe2x80x94OH 
By way of illustration, the method for synthesis in the liquid phase comprises successive condensation, two by two, of the aminoacyls in the required order or condensation of the aminoacyls and fragments which have been formed beforehand and already comprise several aminoacyls in the appropriate order, or several fragments previously prepared in this way, it being understood that it will be necessary to protect all the reactive functions carried by these aminoacyls or fragments beforehand, with the exception of the amine functions of the one and carboxyl functions of the other, or vice versa, which should usually involved in the formation of peptide bonds, in particular after activation of the carboxyl function, in accordance with the methods well-known in the synthesis of peptides.
For example, it would be possible to use protective groups of the urethane type (Boc, Fmoc, benzyloxycarbonyl or allyloxycarbonyl) to protect the N-terminal ends of the amino acids and groupings of the ester type (methyl, ethyl, benzyl, tert-butyl, allyl or benzhydrylglycolamide) to protect the C-terminal ends of the amino acids.
Such a synthesis can be carried out by first condensing the aminoacyl residue AA1, the COOH function of which is protected, with the aminoacyl residue AA2, the NH2 function of which is protected. The amine function of the AA2 residue in the AA2xe2x80x94AA1 fragment thus obtained is then deprotected, for subsequent condensation of the said fragment with the aminoacyl residue AA3, the amine function of which is protected. The preceding stages are repeated for as many times as there are aminoacyl residues to be introduced into the chain of the retro analogues to be synthesized.
According to another preferred technique of the invention, that described by R. M. Merrifield in the article entitled xe2x80x9cSolid phase peptide synthesisxe2x80x9d (J. Am. Chem. Soc. (1963), 85, 2149-2154) is used.
To prepare a peptide chain by the Merrifield process, a highly porous polymeric resin on which the first C-terminal amino acid (in this case AA1xe2x80x94OH) of the chain is fixed is used. This amino acid is fixed on the resin by the intermediary of its carboxyl group, and its amine function is protected, for example by the t-butyloxycarbonyl group.
When the first C-terminal amino acid is fixed on the resin in this way, the protective group of the amine function is removed by washing the resin with an acid.
The amino acids which will make up the peptide chain are thus fixed, one after the other, onto the amino group, deprotected beforehand, of the portion of the peptide chain already formed, which is attached to the resin.
When all the desired peptide chain has been formed, the protective groups of the various amino acids which make up the peptide chain are removed and the peptide is detached from the resin, for example with the aid of hydrofluoric acid.
The preparation of cyclic retro peptides is advantageously carried out in accordance with the method of A. Kates et al., Tetrahedron Lett., 34, 4709, (1993).
If all the starting aminoacyl residues AA1 to AAn used for synthesis of the retro analogues have the same chirality as these same aminoacyl residues which make up the parent peptide, the retro analogue obtained will be a totally retro peptide.
Conversely, if all of these residues AA1 to AAn are of opposite chirality to that of these same residues in the parent peptide, the retro analogue obtained will then be a retro-inverso peptide.
If one or more of, but not all, these residues are of opposite chirality to that of these same residues in the parent peptide, the retro analogue obtained will then be a totally retro-partly inverso peptide.
The preparation of partly retro peptides or partly retro-inverso peptides and also of totally retro or retro-inverso peptides with modification of the ends is advantageously carried out with the aid of the conventional technique using gem-diaminoalkyl residues and substituted C-2 derivatives of malonic acid, described in particular in the article by Pallai and Goodman (Pallai, P. V., and Goodman, M., (1982), J. Chem. Soc. Chem. Commun., 280-281), and in the chapter by Cope et al. ((1957), Org. React., 9, 107) respectively.
In this last reference, the preparation of substituted C-2 derivatives of malonic acid is carried out by alkylation of a diester of malonic acid.
The substituted C-2 monoester of malonic acid can also be obtained by alcoholysis of derivatives of Meldrum acid (Junek et al., (1976), Synthesis, 333-334; Chorev et al., (1983); J. Med. Chem. 26, 129-135), these derivatives themselves being obtained by reductive alkylation of Meldrum acid in accordance with the method described by Hrubowchak, D. M., and Smith, F. X. (1983), Tetrahedron Lett., (24), 4951-4954).
As regards the synthesis of gem-diaminoalkyl derivatives described by Pallai and Goodman, the optically pure mono-N-acylated gem-diaminoalkyl residues are obtained from N-protected amide peptides or from carboxamide aminoacyl derivatives via a Hoffman rearrangement using a moderately oxidizing reagent: iodobenzene bis(trifluoroacetate), called IBTFA or TIB. Furthermore, this method can be used in the solid phase in accordance with the technique described by Pessi et al. (1983), J. Chem. Commun., 195-197.
By way of illustration, the preparation of a retro peptide of the formula:
. . . xe2x80x94NHxe2x80x94CH(R1)xe2x80x94COxe2x80x94NHxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94COxe2x80x94NHxe2x80x94CH(R5)xe2x80x94COxe2x80x94NHxe2x80x94CH(R6)xe2x80x94COxe2x80x94 . . . 
can be carried out by condensation of the amino acid derivative Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COOH, P representing a protective grouping of the urethane type, with the carboxamide derivative H2Nxe2x80x94CH(R3)xe2x80x94COxe2x80x94NH2, followed by treatment of the amide dipeptide obtained with IBTFA, which leads to a mono-N-acylated gem-diaminoalkyl residue of the formula Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NH2, and then condensation of this derivative with a C2-substituted monoester of malonic acid of the formula HOOCxe2x80x94CH(R4)xe2x80x94COOR, xe2x80x94R representing a protective grouping such as xe2x80x94CH3, xe2x80x94CH2CH3, xe2x80x94C(CH3)3 or benzyl, which leads to a fragment of the formula:
Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94COOR, 
followed by selective deprotection of the R grouping, and condensation of the fragment of which the C-terminal acid function is deprotected in this way with a fragment of the formula H2Nxe2x80x94CH(R5)xe2x80x94COxe2x80x94NHxe2x80x94CH(R6)xe2x80x94CO . . . , obtained by condensation of an amino acid derivative Pxe2x80x2xe2x80x94NHxe2x80x94CH(R5)xe2x80x94COOH, Pxe2x80x2 representing a protection of the urethane type, and a fragment of the formula H2Nxe2x80x94CH(R6)xe2x80x94CO . . . , the C-terminal end of which is protected, and selective deprotection of Pxe2x80x2, which leads to a fragment of the formula:
Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94COxe2x80x94NHxe2x80x94CH(R5)xe2x80x94COxe2x80x94NHxe2x80x94CH(R6)xe2x80x94COxe2x80x94 . . . , 
followed by selective deprotection of the protective group P, and condensation of the fragment of which the N-terminal amine function is deprotected in this way with a fragment or a derivative of an amino acid of the formula . . . xe2x80x94NHxe2x80x94CH(R1)xe2x80x94COOH, the N-terminal end of which is protected, which leads to the desired derivative of the formula:
. . . xe2x80x94NHxe2x80x94CH(R1)xe2x80x94COxe2x80x94NHxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94COxe2x80x94NHxe2x80x94CH(R5)xe2x80x94COxe2x80x94NHxe2x80x94CH(R6)xe2x80x94COxe2x80x94 . . . 
containing a gem-diaminoalkyl residue carrying R3 and a derivative of malonic acid carrying R4.
If the production of several successive xe2x80x94NHxe2x80x94COxe2x80x94 bonds within the same partly retro peptide or retro-inverso peptide is desired, it is sufficient to incorporate one or more aminoacyl residue(s) of identical or opposite chirality to this (these) aminoacyl residue(s) in the parent peptide, between a gem-diaminoalkyl residue and a derivative of malonic acid as described above, by the conventional techniques in peptide synthesis.
By way of example, the fragment of the formula:
Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NH2, 
preparation of which by treatment of the amide dipeptide with IBTFA has been described above, can be condensed with an aminoacyl derivative of the formula Pxe2x80x2xe2x80x94HNxe2x80x94CH(R4)xe2x80x94COOH, which leads to the preparation of a fragment of the formula Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94NHxe2x80x94Pxe2x80x2, which, after selective deprotection of the Pxe2x80x2 grouping, can be condensed with a C2-substituted monoester of malonic acid of the formula HOOCxe2x80x94CH(R5)xe2x80x94COOR, xe2x80x94R representing a protective grouping such as xe2x80x94CH3, xe2x80x94CH2CH3, xe2x80x94C(CH3)3, xe2x80x94CH2xe2x80x94CHxe2x95x90CH2 or benzyl, which leads to the fragment of the formula
Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3 )xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94NHxe2x80x94COxe2x80x94CH(R5)xe2x80x94COOR, 
which, after selective deprotection of the R grouping and condensation of the fragment, of which the C-terminal acid function is deprotected in this way, with a fragment of the formula H2Nxe2x80x94CH(R6)xe2x80x94COxe2x80x94 . . . , leads to the fragment of the formula:
Pxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94NHxe2x80x94COxe2x80x94CH(R5)xe2x80x94COxe2x80x94NHxe2x80x94CH(R6)xe2x80x94COxe2x80x94 . . . , 
which, after selective deprotection of the group P, can be condensed with the fragment . . . xe2x80x94NHxe2x80x94CH(R1)xe2x80x94COOH, the N-terminal end of which is protected, which leads to the derivative of the formula
. . . xe2x80x94NHxe2x80x94CH(R1)xe2x80x94COxe2x80x94HNxe2x80x94CH(R2)xe2x80x94COxe2x80x94NHxe2x80x94CH(R3)xe2x80x94NHxe2x80x94COxe2x80x94CH(R4)xe2x80x94NHxe2x80x94COxe2x80x94CH(R5)xe2x80x94COxe2x80x94NHxe2x80x94CH(R6)xe2x80x94COxe2x80x94 . . . 
As above, the preparations of partly retro or retro-inverso peptides by the method described above is determined by the choice of the chirality of the aminoacyl residues which will be capable of being bonded to at least one of their neighbours by a bond of the xe2x80x94NHxe2x80x94COxe2x80x94 type in the analogue obtained.
The invention also relates to the processes for the preparation of totally retro, totally retro-inverso or totally retro-partly inverso immunoretroids as described above, the N- and C-terminal ends of which are modified to mimic further the ends of the parent peptide.
In fact, as we have seen in the preceding synthesis examples starting from the parent peptide represented by the formula:
H2Nxe2x80x94CH(R1)COxe2x80x94NHxe2x80x94CH(R2)xe2x80x94CO . . . NHxe2x80x94CH(Rnxe2x88x921)xe2x80x94COxe2x80x94NHxe2x80x94CH(Rn)xe2x80x94COOH, 
the retro peptide obtained after reversal of all the xe2x80x94COxe2x80x94NHxe2x80x94 bonds is represented by the formula:
Axe2x80x94CH(R1)NHxe2x80x94COxe2x80x94CH(R2)xe2x80x94NH . . . xe2x80x94COxe2x80x94CH(Rnxe2x88x921)xe2x80x94NHxe2x80x94COxe2x80x94CH(Rn)xe2x80x94B 
in which A and B represent, respectively, HOOCxe2x80x94 and xe2x80x94NH2, or any other group initially situated in the N- or C-terminal position of the parent peptide if the analogue is constructed without modification of its ends.
However, it is possible to modify the N- and C-terminal groups of the retro analogue to mimic the N- and C-terminal ends of the parent peptide.
In particular, it is possible to obtain retro analogues of the formula indicated above, in which A represents Hxe2x80x94, H2Nxe2x80x94, Pxe2x80x94HNxe2x80x94, RRxe2x80x2Nxe2x80x94, H2NCOxe2x80x94, RRxe2x80x2NCOxe2x80x94 or RCOxe2x80x94 and B represents xe2x80x94H, xe2x80x94COOH, xe2x80x94COOR, xe2x80x94CONH2-, xe2x80x94CONRRxe2x80x2 or xe2x80x94NHCOR, R, Rxe2x80x2 and P being defined as indicated with regard to the formula (II) above.
By way of illustration, the preparation of retro analogues for which A represents H2Nxe2x80x94 and B represents xe2x80x94COOH can be carried out by incorporating a gem-diaminoalkyl derivative (H2Nxe2x80x94CH(R1)xe2x80x94NHxe2x80x94) to mimic the N-terminal end of the parent peptide and by incorporating a C2-substituted derivative of malonic acid (xe2x80x94COxe2x80x94CH(Rn)xe2x80x94COOH) to mimic the C-terminal end of the parent peptide, by the methods indicated above.
The preparation of retro analogues in which A represents H2NCOxe2x80x94 and B represents xe2x80x94NHCOR, R corresponding to the definition given above, can be carried out by amidation of the COOH of the N-protected amino acid HOOCxe2x80x94CH(R1)xe2x80x94NHxe2x80x94P and by acylation of the N-terminal end; (by way of example, an acetylation is carried out with acetic anhydride in the presence of DIEA [DIEA=diisopropylethylamine], which gives for B: xe2x80x94NHCOCH3).
By combining the various techniques described above for introduction of groups A and B, it is possible to obtain all possible combinations of the groups A and B at the ends of the analogues of the invention.
In this respect, particularly preferred retro analogues in the context of the invention are those for which A represents H2NCOxe2x80x94 and B represents xe2x80x94COOH, obtained by introducing a carboxamide termination into the C-terminal of the retro analogue, and by introducing a C-2-substituted derivative of malonic acid at the N-terminal end of the retro analogue, by the techniques described above.
It is appropriate to state that the malonic acid derivatives are incorporated in the racemic form (written xe2x80x94m(R,S)AAixe2x80x94), and if a chiral centre is to be maintained at the level of this malonate in the peptide, it is necessary to purify the mixture of the two diastereoisomers thus obtained at the end of the synthesis, in particular by liquid phase chromatography.